Mechanical perturbations of myotubes in cultures will be performed by deformation of a flexible substrate, and on rat hind limbs by suspension and reloading. The NOS-dependent responses that will be assayed include changes in expression of structural proteins enriched at MTJs (talin) or regulatory proteins that are enriched at MTJs (NOS). NOS dependence of early genes in response to modified muscle loading will also be tested as will NO-mediated regulation of calcium-dependent proteases that are enriched at MTJs. The specific mechanism by which NO covalently modifies skeletal muscle actin will be examined. Hind limb immobilization at shortened length followed by remobilization will be used to examine the effect of inhibition of NOS on sarcomere addition.